Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
  • A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/42—Gynaecological or obstetrical instruments or methods
  • A61B17/425—Gynaecological or obstetrical instruments or methods for reproduction or fertilisation
  • A61B17/435—Gynaecological or obstetrical instruments or methods for reproduction or fertilisation for embryo or ova transplantation

Definitions

• This invention relates to an in vitro method for the induction of the spermatozoal acrosome reaction and to the application of said method in artificial insemination and in vitro fertilisation (IVF) .
• spermatozoa The vast majority of ejaculated spermatozoa (sperm) are not immediately capable of achieving fusion with an ovum. In the normal course of events, the spermatozoa attain this competence while ascending the female reproductive tract, in particular, in the uterus and the fallopian tubes. The spermatozoa first of all undergo a. reversible capacitation step followed by an irreversible acrosome reaction. In in vitro fertilisation techniques, the acrosome reaction is obtained by a number of routes.
• a spermatozoan is a highly differentiated cell whose role is the protection, transport and delivery into the ooplasm of the male genome.
• the structure of the mammalian spermatozoan varies widely from species to species, however a general mammalian structure is apparent:
• the spermatozoan comprises a head portion linked to a tail portion by a fragile neck portion.
• the tail portion is responsible for the motility of the spermatozoan.
• the spermatozoan head can be divided into two parts, the nucleus and the surrounding membrane structures.
• the membrane structures include the plasma membrane, that covers the entire structure of the head, the acrosome, a bag like structure that surrounds the anterior portion of the nucleus, a postnuclear cap that covers the posterior portion of the nucleus and the equatorial segment which represents the area of overlap between the postnuclear cap and the acrosome.
• the acrosome consists of an inner acrosomal membrane and an outer acrosomal membrane which is in close contact with the plasma membrane.
• the reversible capacitation step enables sperm to penetrate the acellular glycoprotein layer, the zona pellucida, of the ovum (Bedford, J.M. , 1970, Biol.
• the acrosome reaction is a morphological event where the plasma and the outer acrosomal membranes undergo multiple point fusion (Green, D.P.L. , 1978, The mechanism of the acrosome reaction, in Development in mammals, Ed., Johnson, M.H., Vol. 3, 65-81, Nth. Holland, Amsterdam) , resulting in the release of acrosomal hydrolases (Akruk, S.R. et al., 1979 Gamete Res., 2., 1-3).
• a basic assumption is that elevation of Ca ions in the cytoplasm between the plasma and outer acrosomal membranes is the key event just prior to initiation of the acrosome reaction. Capacitation involves the preparation of the sperm for the elevation
• a viable acrosome reaction is an essential prerequisite for sperm fusion with the egg oolemma in mammalian fertilisation.
• SPA sperm penetration assay
• This assay assesses the ability of capacitated human sperm to penetrate surrogate zona-free hamster eggs; quantitative estimates of % penetration, polyspermy and attachment are produced.
• the technique is not standardised, but does nevertheless give good predictive ability of samples' in vivo fertilizing potential, particularly if supported by motility data. Using this system, along with other tests, some categories of patients attending an infertility clinic can be ranked with some accuracy according to fertility potential.
• the SPA has essentially been confined to assessment of infertility in humans to date, although there is a demand at present for extension of the assay for use in assessment of infertility in a number of domestic animals.
• fusion of the zona-free hamster egg has been observed with sperm from the following species: budgerigar, bat, dolphin, mouse, deer mouse, rat, guinea pig, rabbit, dog, pig, goat, bull, horse and marmoset monkey.
• Electropermeabilisation or ele ⁇ troporation is a technique involving use of an electric field pulse of high intensity to generate transient pores in cellular membranes. This technique has been used inter alia to introduce biologically active foreign genes into primary rat hepato ⁇ ytes.
• the invention provides a method of inducing the acrosome reaction in spermatozoa, which comprises subjecting spermatozoa in vitro to electro ⁇ permeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of ca ++ ions, such that up to 100% penetration is observed in the so- called sperm penetration assay (SPA) with normal sperm and up to 75% penetration observed in said assay in oligospermic samples.
• SPA sperm penetration assay
• the invention also provides a method of assessing the developmental potential of spermatozoa, which comprises subjecting spermatozoa in vitro to electro ⁇ permeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 V to +1 V to allow an influx of Ca ions, and determining the ability of the spermatozoa so-treated to penetrate surrogate zona-fre ⁇ hamster eggs, thereby indicating the developmental potential of said spermatozoa.
• the invention further provides a method of preparing spermatozoa for in vitro fertilisation, which comprises inducing the acrosome reaction in said spermatozoa in vitro by subjecting the spermatozoa to electro ⁇ permeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of Ca ions.
• the invention further provides a method of treating male infertility, which comprises converting otherwise non- viable spermatozoa to viable spermatozoa by subjecting said spermatozoa in vitro to electropermeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of Ca ions, thereby inducing the acrosome reaction in said spermatozoa.
• the invention further provides a method of diagnosing male-related infertility which comprises subjecting a sample of spermatozoa in vitro to electro ⁇ permeabilisation involving application of an electric field sufficient to raise ch ⁇ spermatozoal plasma membrane potential from about -70 V to +1 V to allow an influx of Ca ++ ions and determining the ability of the spermatozoa so treated to penetrate surrogate zona-free hamster eggs, thereby indicating the developmental potential of the spermatozoa.
• the methods according to the invention effectively side step all the preparatory capacitation stages by directly permitting calcium ion entrance to a large number of cells inducing rapid and synchronised acrosome reactions.
• cells are subjected to a large and transient DC field such that the plasma membrane resting potential is raised from about -70 V to +1 V, localised regions of the membrane break down and pores are realised.
• the number, size and duration of these pores is a function of the applied voltage, duration of pulse and pulse medium, temperature, as well as, other factors such as cell size and the cholesterol/phospholipid ratio in the membrane.
• the induction of the acrosome reaction in accordance with the invention which brings about direct and rapid influx of Ca ++ ions into sperm, probably bypasses all or some of the membrane changes associated with the strict capacitation phase, i.e., it creates a large and relatively instant population of sperm in various stages of acrosome induction, some of which retain otility.
• the end result of ionophore(4) exposure is similar to electropermeabilisation, but the mechanism is different. Ionophore effectively transports calcium ions through the membrane. Electropermeabilisation would seem to be more effective, yet much less deleterious than ionophore, which usually kills more than half the cells, is difficult to control and does not appear to benefit the majority of sub-fertile samples. All other previous pre-treatment systems generally only induce acrosome reaction in a small percentage of the sperm.
• the electric field is applied in the methods according to the invention as a pulse at a voltage of 250-1000 V corresponding to a field strength of 625-2000 V cm " , especially 400-800 V corresponding to a field strength of 1000-2000 V cm "1 .
• the pulse preferably either emanates (i) from the discharge of a capacitor, (ii) from the variable switching of a static power-supply or (iii) by means of a high voltage pulse generator.
• the former approach gives rise to a pulse which is exponential in character, the latter two approaches will both give relatively clean square pulses of defined shape and duration. Additionally, one could amplify the output from a standard pulse - generator or stimulator.
• a Bio-Rad (Trade Mark) Gene Pulsar may be used to generate a pulse which is exponential in character.
• Suitable operating conditions for human spermatozoa have been found to be 500 V at 25 ⁇ F with a time constant of approximately 2.5 mS.
• An inter-electrode distance of 4 mm is used, such that the application of 500 V implies 1250 V/cm.
• penetration remains at 100% for normal samples at 750 V, but polyspermy is decreased, while voltages below 200 V are sub-threshold.
• a Hoefer Instruments Progenetor may be used to generate a square wave pulse. Appropriate operation conditions for the Hoefer Progenetor are 450 V and 10-50 S pulses.
• Electropermeabilisation is usually conducted at +4 C, since the putative pores tend to stay open longer at lower temperatures before resealing. At lower temperatures the threshold of the breakdown potential is raised. Accordingly, in the methods according to the invention, electropermeabilisation is normally carried out at a temperature in the range 20-25°C.
• the basic method is extremely rapid, taking only minutes, while the time from sample arrival to insemination is only approximately one hour, thereby obviating the need to rigidly regulate temperature.
• sperm incorporation in the SPA decreased by 34% when samples were pulsed at +4°C and 49% when pulsed at 37°C.
• the pulse medium includes a source of Ca ions at a concentration in the range 2.0-20 mM, most especially 10 mM.
• the pulse medium may be an ionic, partially ionic or an essentially non-ionic medium.
• a suitable non-ionic medium for use in the method according to the invention is one based on a polyol, especially a monosaccharide or disaccharide, especially sucrose. Advantages of using non-ionic sucrose is that local current heating effects are minimised and pulse strength is increased in capacitance discharge systems. Mannitol is also suitable for such systems and has the added advantage of scavenging hydroxyl radicals. However, Mannitol has been found to be about 20% less effective than sucrose.
• a suitable ionic-based pulse medium has been found with the following composition: 137 mM NaCl, 20 mM HEPES, 5 mM C1, 0.7 mM Na 2 HP0 , 6 mM glucose and 5 mM CaCl 2 .
• a very successful partial ionic medium has been developed with the following formulation: 210 mM inositol, 30 mM KC1, 5 mM HEPES, 100 ⁇ M inositol triphosphate, 2 mM CaCl_ and 1 mg ml BSA. With both of these media the need for a post-pulse centrifugal wash is eliminated and the sample can simply be diluted to the desired concentration, provided this involves a dilution factor of at least X5.
• the advantages of ionic pulse media are best exploited using square wave pulse generators.
• the pulse medium should also include an agent which prevents sticking of spermatozoa to reaction chambers containing it and maintains motility thereof.
• a suitable such agent is a polymer such as polyvinyl alcohol.
• Serum albumin can also be employed, though its chelation properties will ensure that the concentration of available ionized Ca is reduced up to a third.
• post-pulse gamete interaction is conducted in protein containing media such as albumin - based media.
• Fig. 1 illustrates the relationship between calcium concentration in a sucrose based pulse medium and SPA score parameters, mean percentage penetration, and sperm/egg ration measured as the average number of decondensed sperm heads detected in each zona-free hamster egg.
• Plot a represents mean percentage penetration and plot b represents regression of sperm/egg ratio on CaCl_;
• Fig. 2 illustrates the relationship between applied pulse voltage (Gene-Pulsar) , mean total detected acrosome reactions and the SPA score parameters, mean percentage penetrations and mean sperm/egg ratio.
• Curve a. represents mean percentage penetration
• curve b represents mean total percentage and partially acrosome reacted sperm
• plot ⁇ represents mean sperm/egg ratio
• Fig. 3 illustrates the relationship between postpulse incubation time, percentage penetration, and sperm incorporation.
• Plot a indicates mean percentage penetration and plot b represents mean sperm/egg ratio.
• the s/e ratio is a more sensitive measure of 'fertility' than the other parameters measured in the SPA.
• Semen samples were obtained from patients referred from the Infertility Clinic of Galway Regional Hospital. The patients were a fairly homogenous group with only 20% exhibiting apparent secondary infertility. In 15% of couples, the spouse also demonstrated a reproductive problem. The mean age of the group was 33.5 ⁇ 0.7 years while the mean duration of infertility was 4.3 ⁇ 0.54 years. Approximately 25% showed an abnormal spermiogram on first referral, with the bulk of the remainder being- classified as unexplained infertility. The control group was smaller (N 5) with a mean age of 37.2 ⁇ 3.2 years. All had fathered a child within the previous ten years, had a normal spermiogram and no clinical indications of ill health.
• Semen samples were obtained by masturbation into a sterile Sterilin (Sterilin is a Trade Mark) 60 ml container and kept warm until delivered to the laboratory.
• sterile Sterilin Steerilin is a Trade Mark
• HEPES CaCl 2 .2H 2 0, 0.28 M glucose and 200 mM HEPES, pH 7.3-7.4 (osmolality 360 mOsm, density 1.10 g ml- ) .
• HEPES was prepared as a 1 M stock solution by mixing tissue culture grade acid and base solutions at 37°C to a stable pH of 7.3-7.4. The final osmolality was 312 mOsm.
• the HEPES stock solution was diluted as necessary • to give the desired molarity.
• the upper layer was composed of 47.5% Percoll, 2.5% HEPES buffered saline in complete medium (osmolality 335 mOsm, density 1.05 g ml " ) .
• the gradient was slightly hyperos otic for the reasons described by Vincent, R. and Nadeau D. , (1984) Anal. Biochem. 141. 322-328.
• the loaded gradient was centrifuged at 350 g for 25 minutes in an angle head rotor at room temperature.
• the diffuse sperm pellet at -360 mOsm was then carefully removed, resuspended in 10 ml of fresh medium (freshly prepared medium of the same formulation) as above and 1 ml of Lipiodol (Lipiodol is a Trade Mark for a radio-opaque oil of May & Baker Limited) and twice centrifuged in a swing-out rotor at 350 g at room temperature.
• BWW (herein referred to as BWW/n) is a modified Tyrodes medium, the Biggers, Whitten & Whittinghams medium (Biggers et al., 1971).
• BWW/DM is the defined medium containing a combination of polyvinyl alcohol (PVA) and dextran (Tomkins, P.T. et al., 1988 supra).
• PVA polyvinyl alcohol
• dextran Tomkins, P.T. et al., 1988 supra.
• the complete formulation for BWW/DM is given in Table 1.
• the sperm were suspended at a concentration of 0.5 x 10 8 ml—1 in a pulse medium consisting of 0.26M Analar sucrose, 10 mM Analar
• BWW/DM has proved superior to BWW/n containing 0.3 - 0.5% human serum albumin for inducing acrosome reactions and fusiogenic sperm by means of pre-incubation, it appears to be inhibitory after sperm have been pulsed.
• BWW/DM increases cell leakiness and the post-pulse situation does not adequately preserve cell integrity; motility certainly declines more in BWW/DM than in BWW/n under post-pulse conditions.
• the complete formulation for BWW/n is given in Table 1.
• the group designated *5 hr cap/HSA' in Table 2 was the result of pre-incubating sperm in BWW/n containing 0.5% human serum albumin (HSA) , prior to running in the SPA as usual.
• the group designating '24 hr cap/HSA' in Table 2 was the result of pre-incubating the sperm overnight for 18-24 hours in BWW/n containing 0.3% HSA.
• Control sperm concentrations ranged from 8 x 10 7 ml-1 to 2.5 x 10 8 ml—1, while oligospermic sample concentrations ranged from 1 x 10 6 ml "1 to 1.5 x 10 7 ml -1 .
• Fig. 1 demonstrates the effect of Ca ion concentration of the medium on the electropermeabilisation of sperm in accordance with the invention as measured in terms of s/e ratio and % penetration according to the SPA.
• Membrane vesicular fusion events and the release of hydrolyti ⁇ enzymes during the acrosome reaction are not instantaneous processes. While rapid Ca influx may synchronise acrosome induction in the majority of cells, it is possible that the kinetics differ among subpopulations of sperm. Manipulation of postpulse incubation time was used to investigate this possibility. The mean results of two experiments are shown in Fig. 3. Penetration responses markedly declined if post-pulse incubation at 37 C was continued beyond 1 hour. This may reflect impending senescence of an active acrosome reacted population. Under tube incubation conditions, percentage otility was 72 ⁇ 8% after the
• the SPA is usually held to measure a sperm's ability (and hence the sample) to undergo capacitation, the acrosome reaction, fuse with and be incorporated into an egg and permit DNA decondensation.
• the results of Tables 2 and 3 indicate the methods according to the invention elevate penetration in the SPA. Accordingly, it is expected the basic method according to the invention will prove useful for use as an adjunct to this in vitro test by enabling grouping and class separation on the basis of distinct s/e data, as well as, establishing the ultimate fusiogenic potential of a sample.
• the SPA is now routinely conducted in a great many medical centres but interpretation and cross-evaluation has been hampered by the lack of standardisation in methodology and a relatively high associated level of false-negative responses in the text system (see Tomkins, P.T. et al.. supra 1988) for a discussion of the problem.
• Application of the present invention to the SPA results in a simple and efficient methodology that enables each sperm sample to achieve its maximal and optimised response in the test assay. This increases the resolution of the assay's discriminative powers and can potentially isolate a genuine population of males that do demonstrate pathological failings of the fusiogenic apparatus in all or a proportion of their sperm cells.
• the basic method according to the invention can be used for all in vitro capacitation procedures prior to gamete interaction.
• the basic method according to the invention can also be used in the extension of the SPA known as the sperm chromosome assay (SCA) which establishes the level of normal sperm karyotypes.
• SCA sperm chromosome assay
• the electropermeabilisation method according to the invention can also be used to achieve benign electropermeabilised entry of compounds, in which some sub-fertile sperm may be deficient such as cAMP, taurine and superoxide dismutase, into sperm.
• some sub-fertile sperm may be deficient such as cAMP, taurine and superoxide dismutase, into sperm.
• human sperm from known fertile individuals do not appear to exhibit high levels of acrosome induction following ⁇ apacitative pre-incubation treatments (C.E. Stock & L.R. Fraser 1987, Hum. Reprod. 2, 109-119).

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• 1988
  • 1988-09-22 IE IE872567A patent/IE872567L/xx unknown
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  • 1988-09-23 EP EP88908291A patent/EP0380536A1/de not_active Withdrawn
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